In this study, a three-dimensional cell culture model was applied to study the biological interaction between bone and implants on the cytological scale. It reveals at this in vitro level that the rough surface created by the modified sandblasting surface treatment can induce a real perpendicularly connecting bone-fiber osseointegration that might favor the interfacial biomechanics of implants and at the same time enhance the functions of osteoblasts.

Dental implants have been developed into a scientifically and clinically accepted restoration modality in completely and partially edentulous patients. This achievement has been primarily founded on the principle of osseointegration, which was mainly discovered and defined by Branemark and his colleagues. Attempts have been made to enhance osseointegration by the surface modification of dental implants. The common realization has been that rough surfaces favor osseointegration, and this has led to a variety of implant products with various surface types, such as titanium plasma spray, hydroxyapatite coating, sintered titanium coating, etc. As an alternative noncoating course of study, a modified sandblasted surface has been developed. It features two levels of rough topographic textures (the sandblasted macrotexture and the inhabiting acid etched micropores), no heteroelement pollution on the surface, and no compromise of anticorrosiveness caused by the increasing surface area. It was biologically demonstrated that this new surface could accelerate the initial bone healing process at the implant-bone interface and quadruple interfacial shear strengths. Except for the difference in processing, these results were consistent with the findings of Buser et al with the sandblasted and acid-etched surface that they developed. All these studies were geared to reveal that topography, especially on the scale of cellular dimensions, could work as a biocompatibility-influencing factor as well as a biomechanical factor. Concerning the mechanism, there is no definite answer. Based on cytological experiments, Martin et al and Boyan et al found that topography could influence protein synthesis and differentiation of osteoblasts. Although their findings cast a light on the mechanism of topographic influences on the bone-healing process, the extrapolation was literally restricted by their experimental models, which fell into a two dimensional scope. As a matter of fact, the cells around inserted implants are accommodated in an extracellular fibrils-constructed skeleton. In this study, a three-dimensional experimental cell culture model developed by the authors16 was used to demonstrate the influence of the modified sandblasted surface on the cytological interaction at the implant bone interface. The study was aimed at verifying its feasibility and advantages as an implant surface and revealing its mechanism in influencing bone interface at an in vitro level.

Materials and methods.

Experimental Groups and Preparation of Samples.Eight Ti discs (diameter, 2.5 mm; central hole diameter, 1.0 mm) were cut from grade 2, commercially pure titanium rods (ASTM F67 Unalloyed titanium for surgical implant applications, Northwest Nonferrous Metal Institute, Xian, P.R.China). They were divided into two groups: the modified sandblasted surface group and the smooth surface group. Discs in the former group were sandblasted with 0.15- to 0.21-mm corundum (AL2O3) on circumferential surfaces and modified by oxalic acid attack. The circumferential surface of the latter group was polished through a series of silicon carbide papers to 800-grit. Before use, degreasing of all discs was performed by cleaning in trichloroethylene and ethanol under ultrasonics for 10 minutes each and passivation was performed by immersing in 40% nitric acid for 30 minutes. They were then rinsed with distilled water five times and finally sterilized by autoclaving at 120°C for 30 minutes.

Scanning Electron Microscopy of Ti Disc Circumferential Surface.The topography of Ti-disc circumferential surfaces was observed by scanning electron microscopy (S-520, Hitachi Co., Japan) on one sample from each group after samples were cleaned, passivated, and sterilized.

Cell Culture.Primary osteoblast-like cells were derived from human fetal calvaria following the explant method described by Li et al. Calvaria were isolated from six-month-old human fetuses after legal abortion and washed in Dulbeco modified Eagle medium (DMEM) (Sigma Chemical, St. Louis, MO) supplemented with 300 U/mL penicillin and 300 U/mL streptomycin. After scraping of the periosteal sides and removal of sutures, calvarial plates were cut into small pieces and then plated into flasks cultured with DMEM containing 10% fetal bovine serum, 50 mg/mL L-ascorbic acid at 37°C, and 5% CO2. The medium was changed the next day and every three days thereafter. When reaching confluence at the 12th culture day, the outgrowth cells were subcultured with 0.25% trypsin. They were identified as osteoblast-like cells and well characterized (Fig. 1). The seventh passage osteoblasts were seeded into three wells of a six-well plate in DMEM supplemented with 10 nmol/L dexamethasone and 10 mmol/L Naglycerophosphate as well as 10% fetal bovine serum and 50 mg/mL L-ascorbic acid as above. Until the seventh culture day, the osteoblasts had been reaching confluence.

Plating of Ti Discs.When the confluent cellular layer had formed, the six prepared Ti discs (three per group) were carefully put onto it at an interval of 1 cm; both groups within the same well and the triplicate samples in separate wells. The cells were fed every three days. On each application, intensive care was taken to prevent discs from moving.

Follow-up Observation with Inverted Phase-Contrast Microscope.The culture lasted one month after disc plating. On the first day, cells in the cellular layer migrated, attached, and oriented to the circumferential surfaces of Ti discs. As the culture continued, the migrating and attaching cells increased and a three-dimensional interface between discs and osteoblasts was formed. These events were observed and followed in an inverted phase-contrast microscope (YMT-Z, Olympus Co., Japan). The refractile area surrounding the discs was the portion of interfacial orienting and attaching cells in this three-dimensional situation under the phase contrast condition (original magnification, 340).

Histologic Observation.After the one-month culture, discs associated with the surrounding cells were harvested and processed by rinsing with phosphate-buffered saline, fixing with 4% paraformaldehyde, rinsing again, dehydrating serially with ethanol of increasing concentrations, and embedding in Epon 812. After separation of the embedded surrounding cells from circumferential surfaces of Ti discs by cryofracture and trimming of the derived specimen, they were re-embedded with Epon 812 in a horizontal plane. The specimens were then trimmed again, sliced with ultramicrotome, mounted on a glass slide, and finally stained with methylene blue-basic fuchsin staining method. The sections were 1.0-mm thick. Observation was made with an Olympus microscope (H-2, Olympus Co., Japan) and photographed (Kodak color film 100°, Eastman Kodak, Rochester, NY).

Transmission Electron Microscopy.Specimens of one-month–cultured interfacial cells were processed as above except that the fixation and sectioning were done by the routine method for transmission electron microscopy. The specimens were fixed with 4% paraformaldehyde for 10 minutes, postfixed with 1% osmic acid for 30 minutes, and ultrasectioned with a thickness of 0.02 mm. Observations were made with the JEM-2000EX transmission electron microscope (JEC Co., Japan).

Results.

Scanning Electron Microscopy of Ti Disc Circumferential Surface.The modified sandblasted surface group was distinctive from the smooth surface group with respect to the topographic properties of the circumferential surface. The topography of the smooth surface group was smooth, having only some small parallel scratches (Fig. 2). The modified sandblasted surface, however, was rather rough, but the profile was fairly round. There were numerous secondary micropores inhabiting the rough macrotexture; the average diameter of which was about 2.0 mm. No AL2O3 particles were found embedded in it (Fig. 3).

Follow-up Observation with Phase- Contrast Microscope.After 3 days of culture, discs of the smooth surface group were completely surrounded by a refractile band that appeared uniform in brightness with the cell contours of a parallel orientation and a sharp and regular border. The refractile band in the modified sandblasted surface group was not uniform and had an irregular border. Also, the migrating cells were perpendicular to the Ti disc circumference (Fig. 4). At a high magnification (3400), the refractile band was shown to be a multi-layer structure of cells that oriented themselves in a certain way. In the smooth surface group, cells within this refractile area were mostly attached and parallel to the Ti disc circumference. The attaching cells were long and spindle-shaped. In the modified sandblasted surface group, however, cells attached to the rough Ti disc circumference in a perpendicular manner and the interfacial cells were triangular or multangular (Fig. 5). After 7 days of culture, the attaching and orienting cells increased and the refractile band became broader and brighter. The respective performance of each group was similar to that of the third day, and the differences between the two groups were retained and even expanded.

Histologic Observation.In the horizontal-plane sections, a cell-band arc consisted of tens of cellular layers with the inner side being the Ti-osteoblasts interface with intact histologic structure. The nuclei of cells were oval shaped and stained purplish red, and the cytoplasm was stained pink. The smooth surface group. There were a few cells in this section. Most of them were located opposite the interface. The cell shapes were short spindle or triangle. Long spindle shaped cells with little cytoplasm lined the interface (Fig. 6a). The modified sandblasted surface group. Compared with the smooth surface group, the cell number was larger. Cells of various shapes, mainly round or oval, lined the rough interface. Cytoplasm was abundant, and the nucleus deviated away from the interfacial side. The cells at the interface attached to the rough surface at angles and many collagen-like fibers attached to it in the same way (Fig. 6b).

Ultra-Structure Observation.Apart from the differences in morphology and orientation of interfacial cells demonstrated by ordinary microscopy, the modified sandblasted surface group was also distinguished from the smooth surface group by the morphological phenotypes of cellular function. Compared with the smooth surface group, the modified sandblasted surface was mostly lined by rectangular cells, more abundant in cytoplasm and endoplasmic reticula, and more active in secretion of collagen fibrils (Fig. 7). Furthermore, it was distinguished from the smooth surface by the process of bone matrix mineralization mediated by bone matrix vesicles (Fig. 8) and the formation of osteocytes trapped by mineralized collagen fibrils (Fig. 9). These bone matrix mineralization phenomena were hardly discernible in the smooth surface group.

Discussion - References.

Rough surfaces have been shown to be advantageous to osseointegration. They can provide a better initial stability to dental implants. Additionally and most importantly, they can promote the differentiation of osteoblasts at the bone interface and enhance their osteogenic function. This can accelerate the bone healing process and increase the bone contact rate. These effects of rough surface on the osteogenesis at the bone interface are considered to be functions of its roughness and topography. The modified sandblasted rough surface described herein features the formation of numerous secondary micropores and a rough and round profile. These characteristics happen to be consistent with Wong’s concept concerning an ideal rough surface. He held that, as far as interfacial biomechanics is concerned, the rough surface with more small peaks is better than that with fewer larger peaks. In this in vitro study, the modified sandblasted surface’s biologic effects on the initial healing process at the dental implant-bone interface were investigated by using an experimental three-dimensional model of biomaterial-osteoblast culture developed by the authors. After Ti discs were put into this model, osteoblastic cells migrated from the confluent cell layer to the disc circumference and attached to it, forming a three dimensional interface of osteoblasts to the Ti disc. On the first day, the variance between the two surface groups could not be discerned. By the third day, the effects of the modified sandblasted surface had become apparent. It induced a perpendicular attachment with the triangular or multangular cells at the interface. These were reflected by the nonuniform and broader refractile band under the phase-contrast microscope, having cell contour perpendicular to the disc circumference and reflected by the perpendicularly attaching cells and collagen-like fibers in the cytohistologic sections. Meanwhile, a circular parallel attachment of long spindle-shaped cells occurred in the smooth surface group. Based on this in vitro study, it can be concluded that the real perpendicularly connecting bone-fiber osseointegration can be achieved by the modified sandblasting surface treatment at the dental implant bone interface. The osseointegration of this pattern will possibly enhance the bonding strength of the bone interface and favor the distribution of occlusive force at the interface when compared with the capsule-like adaptation of a smooth surface dental implant. This was also implied by Schroeder’s findings during his study of the histologic effects of titanium plasma spray on the tissue interface of dental implants. He found functionally oriented fibroblastic cells and collagen fibers at the gingival interface that attached to the abutment at angles. This functional connection seemed to increase the bonding strength at the gingival interface. Another distinction in the osteogenic functions of interfacial osteoblasts was revealed by using cytomorphologic methods. Apart from the implication of cell morphology, the active secretion of collagen fibrils, the bone-matrix-vesicles–mediated mineralization, and the formation of osteocytes suggest that the modified sandblasted surface can definitely improve the osteogenic function of interfacial osteoblasts and can be expected to accelerate the process of bone healing at the interface of implants. The same conclusion has been reached by other researchers who have used a two dimensional cell culture model (that is, seeding and culturing cells on designed surfaces). In the present study, the results, based on this more closely in vivo-simulating model, are more significant and more convincing. They confirm the findings of a previous in vivo study and indicate that the modified sandblasted surface accelerates osseointegration through its influence on the morphology and function of interfacial osteoblasts. They also indicate that the surface topography is a factor in biomaterial biocompatibility.